Application of a yeast model in the evaluation of the antioxidant capacity of selected small molecules

To assess the capacity of small molecule antioxidants to protect cells compromised in their antioxidant machinery, yeast strains deficient in catalase A (�cta1), and double deficient in old yellow enzyme 2 and glutathione reductase 1 (�oye2 glr1) containing high levels of oxidized glutathione (GSSG), were subjected to pro-oxidant insult. H2O2 and Cumene Hydroperoxide (CHP) were added to cells growing in complete minimal media (CM) supplemented with ascorbic acid, beta-carotene, caffeic acid, chlorogenic acid, tocotrienols (� and � isomers) or quercetin and monitored for growth recovery. The results show that ascorbic acid, caffeic acid and �� tocotrienol protected cells under most circumstances, whereas beta-carotene, chlorogenic acid and quercetin protection was highly context dependent, exhibiting protection in some cases and inhibition in others. Binary combinations of the most potent compounds ascorbic acid, caffeic acid and ��-tocotrienol at high concentrations 50�M, with the other compounds at low concentrations 5�M in �cta1 treated with H2O2, ascorbic acid (AA) combination with beta-carotene showed the best improvement compared to ascorbic acid at 50�M. In the same purpose of AA combination, but with increasing the doses of ��-tocotrienol (25�M, 50�M and 100�M) in wild type cells treated with H2O2, the results exhibited a proportional relation between survival of cells and concentrations of ��-tocotrienol supplemented. Incubation of cells with beta-carotene and quercetin elevated substantially endogenous levels of reactive oxygen species (ROS). Quercetin supplementation increased significantly GSH and GSSG levels but could not maintain GSH levels in H2O2 exposed cells. Induction of the stress response machinery was manifested by the strong upregulation of a chromosomally encoded OYE2-GFP fusion. In the case of quercetin, supplementation at 50�M caused simultaneous induction of OYE3-GFP. Heterodimer oye2p-oye3p was previously shown to sensitize cells to H2O2-induced programmed cell death (PCD) which is also seen in the growth recovery assay of quercetin treated cells. GFP-YAP1 fusion strain was generated in order to examine the adaptive change of yap1 expression to quercetin supplementation by fluorimetry. The results showed no change in fluorescence intensity compared to the different doses of quercetin supplemented.